Hello everyone,
I have a DNA sample that has been purified from FFPE tissue using the QIAmp DNA FFPE Tissue kit. I obtained a total amount of 1,5 ng of purified DNA, measured with NanoDrop: too little to be used in NGS downstream applications, for which at least 40 ng are required.
I also noticed that commercially available Whole Genome Amplification (WGA) kits usually require at least 10 ng of DNA input, so I’m wondering whether a WGA could be useful to perform a NGS analysis in my case, or whether it won't be suitable.
Do you have experience in WGA starting with a very small amount of material? Alternatively, do you have any advise to properly exploit this sample?
Thank you ;)
Chiara
I have used as little as 500 pg of input DNA into library prep for sequencing. This was from FFPE samples and laser microdissection. Here is the kit I used, the NEB Next ultra II (link below). It is cheaper than the illumina kits, works just as well, and the reaction volumes are HUGE, so you can half them and decrease your input to 250pg and get twice as many reactions out of the kit.
You only really need something like WGA when you are doing single cell sequencing.
Also, I wouldn't trust a nanodrop for concentrations less than 10 ng/microliter. You should use a bioanalyzer or qubit.
https://www.neb.com/products/e7645-nebnext-ultra-ii-dna-library-prep-kit-for-illumina#Product%20Information
hope this link is useful
https://www.nature.com/articles/s41598-017-04419-9
regards
I have used as little as 500 pg of input DNA into library prep for sequencing. This was from FFPE samples and laser microdissection. Here is the kit I used, the NEB Next ultra II (link below). It is cheaper than the illumina kits, works just as well, and the reaction volumes are HUGE, so you can half them and decrease your input to 250pg and get twice as many reactions out of the kit.
You only really need something like WGA when you are doing single cell sequencing.
Also, I wouldn't trust a nanodrop for concentrations less than 10 ng/microliter. You should use a bioanalyzer or qubit.
https://www.neb.com/products/e7645-nebnext-ultra-ii-dna-library-prep-kit-for-illumina#Product%20Information
The problem could come from degraded DNA. I saw that DNA samples from FFPE that had 100s of ng/ul in Nanodrop, had 10s of ng/ul in Qubit, and after library preparation, there is no DNA at all, or not at the right size.
Try running in DNA gel to verify
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